Ji Z S, Fazio S, Lee Y L, Mahley R W
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
J Biol Chem. 1994 Jan 28;269(4):2764-72.
To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.
为了确定增强的载脂蛋白（apo）E分泌对残余脂蛋白摄取机制的影响，将大鼠肝癌细胞（McA-RH7777）用正常人apoE3或受体结合缺陷型apoE-莱顿进行稳定转染。孵育2小时后，转染的肝细胞分泌的人apoE比内源性大鼠apoE高10 - 12倍。与apoE-莱顿转染细胞和未转染细胞相比，apoE3转染细胞结合并内化兔β-极低密度脂蛋白（β-VLDL）的程度要大得多。与apoE-莱顿转染或未转染细胞相比，分泌apoE3的细胞与细胞相关的β-VLDL增强了2 - 3.5倍。观察到荧光标记的β-VLDL集中在apoE3转染细胞的细胞内颗粒中，推测在内涵体和溶酶体内。此外，电子显微镜显示，与未转染或分泌apoE-莱顿的细胞相比，分泌apoE3的细胞在其细胞表面和微绒毛上有丰富的β-VLDL和乳糜微粒残余物。电子显微镜还显示apoE3转染的肝细胞的细胞内囊泡和多囊泡体内有大量乳糜微粒残余物。肝素酶处理（3单位/毫升）完全消除了β-VLDL与apoE3转染细胞增加的结合，但不影响β-VLDL与apoE-莱顿转染或未转染细胞的有限结合。我们确定富含apoE3的β-VLDL与细胞表面硫酸乙酰肝素蛋白聚糖结合，新合成和分泌的apoE3也是如此（约12%的总分泌apoE3被肝素酶和苏拉明释放；4%被肝素释放）。此外，在将β-VLDL与外源性apoE3、apoE3分泌细胞的培养基或apoE3分泌细胞本身孵育后，通过快速液相色谱法重新分离β-VLDL，结果显示这些颗粒富含apoE3并表现出增强的结合。这些结果表明apoE具有分泌捕获作用，并表明细胞表面硫酸乙酰肝素蛋白聚糖在残余脂蛋白代谢中起重要作用。
